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Association of C-Type Lectin Mincle with Fc ε RI β γ Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk

View Article: PubMed Central - PubMed

ABSTRACT

Macrophage-inducible C-type lectin (Mincle) interacts with the γ-subunit of high-affinity IgE receptor (FcεRIγ) and activates Syk by recognizing its specific ligand, trehalose-6,6′-dimycolate, a glycolipid produced by Mycobacterium tuberculosis. It has been suggested that mast cells participate in the immune defense against pathogenic microbes including M. tuberculosis, although the functions are still uncertain. In this study, we examined the Mincle-mediated signaling pathway and cellular responses using RBL-2H3 cells. Mincle formed a protein complex with not only FcεRIγ but also FcεRIβ in a stable cell line expressing myc-tagged Mincle. In addition, engagement of Mincle increased the levels of protein tyrosine phosphorylation and ERK phosphorylation. A pull-down assay demonstrated that cross-linking of Mincle induced binding of FcεRIβγ subunits to the Src homology 2 domain of Syk. Pharmacological and genetic studies indicated that activation of Syk was critical for Mincle-mediated activation of phospholipase Cγ2, leading to the activation of ERK and nuclear factor of activated T cells. Moreover, engagement of Mincle efficiently induced up-regulation of characteristic mast cell genes in addition to degranulation. Taken together, our present results suggest that mast cells contribute to Mincle-mediated immunity through Syk activation triggered by association with the FcεRIβγ complex.

No MeSH data available.


Related in: MedlinePlus

Mincle-mediated expression of characteristic mast cell genes through Syk.Cells expressing WT Mincle were preincubated with or without 2 μM R406 for 5 min (R406) and then stimulated with or without 10 μg/ml anti-myc mAb for 2 h (anti-myc). (a) Heat map of differentially expressed genes (total 1643 genes) was generated using microarray data obtained from the indicated cells in duplicate. Genes with /log2 (fold change)/ > 0.3 and P < 0.05 (ANOVA) were considered to be differentially expressed. See Supplementary Dataset 1 for a full list of selected probe sets and fold changes. Data processing, normalization, statistical analysis, and hierarchical clustering by unweighted pair group method with arithmetic mean (UPGMA) were performed using Subio Platform version 1.18. Expression levels are coloured green for low intensities and red for high intensities. Gene expression including IL-3, IL-4, IL-13, IL-31, CCL1, CCL7, and TNF-α was up-regulated in cells stimulated by anti-myc mAb in the absence of R406. The list of more relevant genes regulated by Syk in Mincle-stimulated RBL-2H3 cells can be found as Supplementary Table S1. The 20 most up-regulated genes are listed in Table 1. (b) Mincle-induced characteristic gene expression was analyzed by quantitative real-time PCR. Data are representative of three independent experiments and are presented as the mean ± S.D. (*P < 0.001 was considered significant. n = 3/group). (c) After cells expressing WT Mincle were stimulated with or without plate-coated TDM for 8 h (TDM), quantitative measurement of IL-4 in cell supernatants or IL-13 in cell lysates was performed by ELISA. Data are representative of three independent experiments and presented as the mean ± S.D. (*P < 0.01 and **P < 0.05 were considered significant. n = 3/group).
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f5: Mincle-mediated expression of characteristic mast cell genes through Syk.Cells expressing WT Mincle were preincubated with or without 2 μM R406 for 5 min (R406) and then stimulated with or without 10 μg/ml anti-myc mAb for 2 h (anti-myc). (a) Heat map of differentially expressed genes (total 1643 genes) was generated using microarray data obtained from the indicated cells in duplicate. Genes with /log2 (fold change)/ > 0.3 and P < 0.05 (ANOVA) were considered to be differentially expressed. See Supplementary Dataset 1 for a full list of selected probe sets and fold changes. Data processing, normalization, statistical analysis, and hierarchical clustering by unweighted pair group method with arithmetic mean (UPGMA) were performed using Subio Platform version 1.18. Expression levels are coloured green for low intensities and red for high intensities. Gene expression including IL-3, IL-4, IL-13, IL-31, CCL1, CCL7, and TNF-α was up-regulated in cells stimulated by anti-myc mAb in the absence of R406. The list of more relevant genes regulated by Syk in Mincle-stimulated RBL-2H3 cells can be found as Supplementary Table S1. The 20 most up-regulated genes are listed in Table 1. (b) Mincle-induced characteristic gene expression was analyzed by quantitative real-time PCR. Data are representative of three independent experiments and are presented as the mean ± S.D. (*P < 0.001 was considered significant. n = 3/group). (c) After cells expressing WT Mincle were stimulated with or without plate-coated TDM for 8 h (TDM), quantitative measurement of IL-4 in cell supernatants or IL-13 in cell lysates was performed by ELISA. Data are representative of three independent experiments and presented as the mean ± S.D. (*P < 0.01 and **P < 0.05 were considered significant. n = 3/group).

Mentions: Next, we performed microarray analysis to identify genes with expression up-regulated by Mincle stimulation in RBL-2H3 cells (Fig. 5a and Table 1). As observed in macrophages and DCs, up-regulation of TNF-α17182021222539 and early growth response transcription factor 1–340 genes was observed in Mincle-stimulated RBL-2H3 cells, whereas that of other reported genes was not observed, such as MIP-2, KC, IL-2, IL-6, and IL-101720222539. In contrast, it appeared that up-regulation of mRNAs encoding IL-3, IL-4, IL-9, IL-13, IL-31, C-C motif chemokine ligand (CCL) 1, and CCL7 was characteristic of RBL-2H3 cells, because those have not been reported in the studies using other cell types. Taken together, these results suggest that the pattern of Mincle-mediated gene expression in RBL-2H3 cells is, at least in part, different from those observed in macrophages and DCs.


Association of C-Type Lectin Mincle with Fc ε RI β γ Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk
Mincle-mediated expression of characteristic mast cell genes through Syk.Cells expressing WT Mincle were preincubated with or without 2 μM R406 for 5 min (R406) and then stimulated with or without 10 μg/ml anti-myc mAb for 2 h (anti-myc). (a) Heat map of differentially expressed genes (total 1643 genes) was generated using microarray data obtained from the indicated cells in duplicate. Genes with /log2 (fold change)/ > 0.3 and P < 0.05 (ANOVA) were considered to be differentially expressed. See Supplementary Dataset 1 for a full list of selected probe sets and fold changes. Data processing, normalization, statistical analysis, and hierarchical clustering by unweighted pair group method with arithmetic mean (UPGMA) were performed using Subio Platform version 1.18. Expression levels are coloured green for low intensities and red for high intensities. Gene expression including IL-3, IL-4, IL-13, IL-31, CCL1, CCL7, and TNF-α was up-regulated in cells stimulated by anti-myc mAb in the absence of R406. The list of more relevant genes regulated by Syk in Mincle-stimulated RBL-2H3 cells can be found as Supplementary Table S1. The 20 most up-regulated genes are listed in Table 1. (b) Mincle-induced characteristic gene expression was analyzed by quantitative real-time PCR. Data are representative of three independent experiments and are presented as the mean ± S.D. (*P < 0.001 was considered significant. n = 3/group). (c) After cells expressing WT Mincle were stimulated with or without plate-coated TDM for 8 h (TDM), quantitative measurement of IL-4 in cell supernatants or IL-13 in cell lysates was performed by ELISA. Data are representative of three independent experiments and presented as the mean ± S.D. (*P < 0.01 and **P < 0.05 were considered significant. n = 3/group).
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Related In: Results  -  Collection

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f5: Mincle-mediated expression of characteristic mast cell genes through Syk.Cells expressing WT Mincle were preincubated with or without 2 μM R406 for 5 min (R406) and then stimulated with or without 10 μg/ml anti-myc mAb for 2 h (anti-myc). (a) Heat map of differentially expressed genes (total 1643 genes) was generated using microarray data obtained from the indicated cells in duplicate. Genes with /log2 (fold change)/ > 0.3 and P < 0.05 (ANOVA) were considered to be differentially expressed. See Supplementary Dataset 1 for a full list of selected probe sets and fold changes. Data processing, normalization, statistical analysis, and hierarchical clustering by unweighted pair group method with arithmetic mean (UPGMA) were performed using Subio Platform version 1.18. Expression levels are coloured green for low intensities and red for high intensities. Gene expression including IL-3, IL-4, IL-13, IL-31, CCL1, CCL7, and TNF-α was up-regulated in cells stimulated by anti-myc mAb in the absence of R406. The list of more relevant genes regulated by Syk in Mincle-stimulated RBL-2H3 cells can be found as Supplementary Table S1. The 20 most up-regulated genes are listed in Table 1. (b) Mincle-induced characteristic gene expression was analyzed by quantitative real-time PCR. Data are representative of three independent experiments and are presented as the mean ± S.D. (*P < 0.001 was considered significant. n = 3/group). (c) After cells expressing WT Mincle were stimulated with or without plate-coated TDM for 8 h (TDM), quantitative measurement of IL-4 in cell supernatants or IL-13 in cell lysates was performed by ELISA. Data are representative of three independent experiments and presented as the mean ± S.D. (*P < 0.01 and **P < 0.05 were considered significant. n = 3/group).
Mentions: Next, we performed microarray analysis to identify genes with expression up-regulated by Mincle stimulation in RBL-2H3 cells (Fig. 5a and Table 1). As observed in macrophages and DCs, up-regulation of TNF-α17182021222539 and early growth response transcription factor 1–340 genes was observed in Mincle-stimulated RBL-2H3 cells, whereas that of other reported genes was not observed, such as MIP-2, KC, IL-2, IL-6, and IL-101720222539. In contrast, it appeared that up-regulation of mRNAs encoding IL-3, IL-4, IL-9, IL-13, IL-31, C-C motif chemokine ligand (CCL) 1, and CCL7 was characteristic of RBL-2H3 cells, because those have not been reported in the studies using other cell types. Taken together, these results suggest that the pattern of Mincle-mediated gene expression in RBL-2H3 cells is, at least in part, different from those observed in macrophages and DCs.

View Article: PubMed Central - PubMed

ABSTRACT

Macrophage-inducible C-type lectin (Mincle) interacts with the &gamma;-subunit of high-affinity IgE receptor (Fc&epsilon;RI&gamma;) and activates Syk by recognizing its specific ligand, trehalose-6,6&prime;-dimycolate, a glycolipid produced by Mycobacterium tuberculosis. It has been suggested that mast cells participate in the immune defense against pathogenic microbes including M. tuberculosis, although the functions are still uncertain. In this study, we examined the Mincle-mediated signaling pathway and cellular responses using RBL-2H3 cells. Mincle formed a protein complex with not only Fc&epsilon;RI&gamma; but also Fc&epsilon;RI&beta; in a stable cell line expressing myc-tagged Mincle. In addition, engagement of Mincle increased the levels of protein tyrosine phosphorylation and ERK phosphorylation. A pull-down assay demonstrated that cross-linking of Mincle induced binding of Fc&epsilon;RI&beta;&gamma; subunits to the Src homology 2 domain of Syk. Pharmacological and genetic studies indicated that activation of Syk was critical for Mincle-mediated activation of phospholipase C&gamma;2, leading to the activation of ERK and nuclear factor of activated T cells. Moreover, engagement of Mincle efficiently induced up-regulation of characteristic mast cell genes in addition to degranulation. Taken together, our present results suggest that mast cells contribute to Mincle-mediated immunity through Syk activation triggered by association with the Fc&epsilon;RI&beta;&gamma; complex.

No MeSH data available.


Related in: MedlinePlus